The goal of chromatography is to separate materials. Chromatography techniques are used for a variety of purposes, from research in basic science to purification of pharmaceuticals. The application of chromatography techniques to production levels works to some degree but when methods and devices are scaled up to the large sizes needed for pharmaceutical or biological product production, most methods and devices work inadequately.
There are many different kinds of chromatography methods and materials. Some of these include paper and thin layer chromatography methods, columns and resins of all types, high pressure liquid chromatography, expanded bed techniques, and reverse phase and reverse flow methods. For example, the first stage in many purification processes of proteins from a fermentation broth, whether from microbial, plant, or animal cell culture, is capture of the desired proteins from the broth. A typical method for accomplishing this is to use an adsorbent material in an expanded bed. On a large scale, the expanded bed uses an upward operating flow through the bed and the flow rate is restricted by increased viscosity, the density of chromatographic adsorbents used, and the rate of binding of the desired protein to the adsorbent. There are also only a few adsorbent materials, such as beads, that can be used due to the presence of only a few types of functional binding groups, particle size, and density of the particle. Additionally, the columns used to perform the chromatography often become contaminated by bacterial or fungal growth, or blocked due to cellular debris. All of these problems lead to a slower process with less material isolated.
The majority of processes for producing pharmaceutical or diagnostic products involve the purification of proteins and peptides from bacteria, yeast and plant or animal cell culture fluids, or extracts from tissues. Usually purification plants use multiple unit operations, including a number of chromatographic steps to ensure the removal of impurities and contaminants. The type of product produced and its intended use will dictate the extent of purification needed. Each step in the recovery process will affect the overall process efficiency by increasing operational costs and process time, and by also causing loss in product yield. Careful selection and combination of suitable unit operations during the design phase may reduce the number of steps needed. The fewest possible processing steps offers the most efficient way of reaching high process efficiency and low costs in the overall production process. Most currently used processes still involve multiple steps of processing which add to the costs, loss of product and offer opportunities for contamination.
Problems in isolation of materials begins in the earliest stages, such as clarification of a fermentation broth or an initial tissue homogenization. Standard techniques for removal of cells or debris are centrifugation and microfiltration. The efficiency of a centrifugation step depends on particle size, density difference between the particles and the surrounding liquid, and viscosity of the feedstock. Although microfiltration may yield cell free solutions, the flux of liquid per unit membrane area is often dramatically decreased during the filtration process. Fouling of the microfiltration membranes is another problem that significantly adds to the operational cost. The combined use of centrifugation and filtration often results in long process times or the use of comparatively large units causing significant capital expenditure and recurrent costs for equipment maintenance. It also results in significant product loss due to product degradation. What is needed are methods, compositions, and devices that allow for direct adsorption from crude feed stocks that can reduce the time and cost of the initial steps of purification.
An alternative to methods of clarification and packed bed chromatography is adsorption to a resin in a stirred tank. This technique is often useful when recovering the target substance from a large volume of crude feed. This method has long been used on a commercial scale for the isolation of plasma coagulation Factor IX with DEAE Sephadex. A major drawback to this system is that well-mixed batch adsorption process is a single-stage adsorption procedure and requires more adsorbent to achieve the same degree of adsorption as in a multi-stage (multi-plate) process such as packed bed chromatography.
A very widely used technique for bulk separation is adsorption of the target molecules in a fluidized bed. This technique can eliminate the need for particulate removal. Fluidized beds have been used in industry for many years for the recovery of antibiotics including batch-processing techniques for recovery of streptomycin and semi-continuous systems for novobiocin. In a fluidized bed, channeling, turbulence, and backmixing is extensive, and is similar to a batch process in a stirred tank. The single equilibrium stage in a fluidized bed decreases the efficiency of the adsorption process with low recoveries, causes the need for re-cycling the media, inefficient washing procedures and increased processing time.
Approaches to solving these problems have been tried by many techniques so that a fluidized bed would have separation characteristics similar to packed bed chromatography. One approach uses segmentation of the bed by insertion of a number of plates with suitably sized holes into the adsorption column. In another approach, magnetic adsorbent particles and a magnetic field over the fluidized bed column are used to stabilize the bed. A substantial stabilization of the bed was achieved using magnetic adsorbents but the experiments were carried out at small laboratory scale and scaling up requires complicated and expensive equipment. Another approach uses agarose in a column equipped with a liquid distribution inlet giving a plug flow in the column.
When these expanded beds were actually used with mixtures of proteins and cells there was some improvement. The breakthrough capacity in such beds, expanded by a factor of two, was very similar to the breakthrough capacity in a packed bed. However, low flow velocities had to be applied to prevent the bed from expanding too much, which resulted in a low overall productivity.
Problems also occur with the particles used for separation. Many standardly used particles are not sturdy enough to withstand the weight of a large column bed, nor can they withstand harsh chemical treatments used for cleaning the beds and columns. The packing materials or resins deteriorate over time due to clean-in-place procedures, harsh buffer conditions, and changing buffer conditions. Additionally, the entire column, piping, or resins may become contaminated, either through bacterial or fungal growth, or through accumulation of material on the particles or resins and that lowers the efficiency. This requires expenditures for replacement of the resins, cleaning all equipment and then assurances that the column has been returned to a good, reliable working condition.
Therefore, a need exists for systems, methods and devices that can separate biomaterials or chemicals that overcome the problems seen with currently used chromatography devices. It is preferred that such systems, methods, and devices be capable of using chromatographical techniques and resins or materials to isolate and separate biomaterials are chemicals more efficiently, and with lower production costs.